1. Field of the Invention
The invention is related to a continuous centrifugation apparatus and method. A continuous flow centrifuge adapted with an insert of glass beads, sand or the like is used to concentrate cysts of microorganisms such as giardia or cryptosporidium from large volumes of water. The apparatus and methods of the invention facilitate the detection of such contaminating organisms in groundwater and public drinking water.
2. Background Information
The problem of human cryptosporidiosis is emerging as one of the more serious issues confronting water purification and distribution facilities both in the United States and elsewhere. Recent outbreaks in Carrolton (Hayes et al., N. Engl. J. Med. 320:1372–1373, 1988), Jackson County, (Leland et al., J. Am. Water Works Assoc. 85:34, 1993) and Milwaukee (Mackenzie et al., 1994 N. Engl. J. Med. 331:161, 1994), have brought matters to a head and in a recent announcement, the US Environmental Protection Agency has required testing of water supplies for oocysts of Cryptosporidium parvum to prevent transmission of the parasite.
Oocysts of C. parvum are common contaminants of natural waterways and have been found in surveys (Ongerth and Stibbs, Appl. Environ. Microbiol. 53:672–676, 1987); Hansen and Ongerth, Appl. Environ. Microbiol. 57:2790–2795, 1991). Outbreaks of cryptosporidiosis appear to be associated with technical aberrations at water treatment facilities or changes in river flow following drought conditions or high level of snow melt. Such conditions may combine to increase the load of C. parvum oocysts in the natural waterways, as well as allow the organisms to break through the filtration purification barrier and get access to human populations. The extent to which the organism occurs naturally has been explained recently in a review by Lisle and Rose (Aqua 44:103–117, 1995) and stresses the gravity of the public health problem.
Isolation and extraction of the oocysts of this parasite from natural water or from water being processed or from finished drinking water is difficult and expensive. Current methods utilize filtration as the main extraction process, but filtration of large volumes of water poses major problems particularly with natural water. Even at low turbidities (NTU values ±5), debris and colloidal material soon blocks the filtration medium making the procedure cumbersome and recovery rates become variable and unpredictable. To overcome this, filtration apparatus become large and require vacuum or pump assistance in order to process the samples. The regular method for the concentration and isolation of protozoal cysts from water utilize spiral wound filters to handle large volumes of water (Proposal P229, Annual Book of the American Society for Testing and Materials, Philadelphia, Pa.). The device requires a pump assisted, pressurized apparatus to extract material from the water sample. The apparatus is recommended for use in clarified water (turbidity ±1 NTU). Filtration of 380 L of water constitutes the sample. Following this the fiber filter unit is removed, the apparatus and filter rinsed with elution fluid, and the filter then slit into shreds with a razor knife, washed, kneaded and finally sonicated in 1 L volumes of elution fluid. The cysts which have been recovered from the water are then removed by elution and concentrated by centrifugation and purified on a Percoll-sucrose gradient. The procedure is cumbersome and leaves several procedures vulnerable to loss of material and hence a source of variability in the results. Reported concentrations of both C. parvum oocysts and Giardia intestinalis cysts from water samples analyzed through current methods have wide confidence limits due to poor and inconsistent recovery (5–40%), a high degree of variability (0–130%) and occurrence of false positives (AWWA: 1996 RFP No 364 “New Approaches for isolation of Crytosporidium and Giardia”).
Use of centrifugation for retrieval of particulate matter from water is well known. However the concept of continuous flow centrifugation has not been used frequently apart from separation of cream from milk. The process differs from previously known continuous flow centrifugation methods in that a column of particulate matter is added to the fluid stream. The matrix (water) containing cysts is added continuously to the centrifuge, and excess water overflows and drains away, while the particles of interest remain trapped in the centrifuge tubes by force of gravity. A system was developed in Zimbabwe by Barrett and Ellison (Cent. Afr. J. Med. 11(11) 338–340, 1965) to separate trematode cercariae from natural waters. The present invention represents a significant advance over the system of Barrett and Ellison.
Extensive field testing demonstrates its superiority compared, for example, with filtration methods and used with considerable success in natural water systems in Egypt (Yousif et al., 1996). The unique operational advantage of this system is that it can handle large volumes of water irrespective of turbidity. It has been shown by Yousif et al., (1996) to operate both in clear water and water with turbidity above 30 NTU and concentrate particulates in the centrifuge tubes even at speeds of 2–3,000 rpm. The cystic stages of both C. parvum and G. intestinalis are much smaller than the trematode larvae and other particles which were isolated by the centrifuge described by Barrett and Ellison, so additional modifications were needed to proceed.